Stabilised phenylalanine ammonia lyase

ABSTRACT

This invention relates to stabilization of phenylalanine ammonia lyase against proteolytic degradation by chemical modification with crosslinking agents, or by genetic modification, a phenylalanine ammonia lyase variant, a method of preparing the variant and a pharmaceutical composition containing phenylalanine ammonia lyase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a PCT/DK94/00224 filed Jun. 9, 1994, which isincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a stabilised phenylalanine ammonialyase, a phenylalanine ammonia lyase variant, a method of preparing thevariant and a pharmaceutical composition containing phenylalanineammonia lyase.

BACKGROUND OF THE INVENTION

Hyperphenylalaninemia, which may be defined as a plasma level ofphenylalanine of more than 120 μmol/l, is a hereditary disease caused bya deficiency in the hepatic enzyme phenylalanine hydroxylase or (in rarecases) its cofactor tetrahydropterin or the cofactor-regenerating enzymedihydropterin reductase. The disease exists in different forms,phenylketonuria (PKU) which, if the patient is on a normal diet, hasplasma phenylalanine levels of more than 1200 μmol/l, and non-PKUhyperphenylalaninemia which has lower levels of plasma phenylalanine.

In normal subjects, phenylalanine hydroxylase converts phenylalanine totyrosine. Highly increased plasma levels of phenylalanine (>600 μmol/L)result in mental retardation. The effect appears to be ascribable tophenylalanine itself (not any metabolites thereof), but the mechanism isnot yet fully understood. In most industrialised countries, newbornchildren are routinely screened for hyperphenylalaninemia. The negativeeffects of increased plasma levels of phenylalanine may, to a largeextent, be prevented if a low-phenylalanine diet is introduced shortlyafter birth and continued well into adolescence. The aim is to obtainplasma phenylalanine levels of 180-425 μmol/l. After adolescence, thelow-phenylalanine regimen may be somewhat relaxed, althoughphenylalanine-free products are still a significant component of thediet. Pregnant hyperphenylalaninemic patients are required to go back ona strict low-phenylalanine diet in order to avoid the effects ofexcessive intrauterine phenylalanine, i.e. congenital malformation,microcephaly and mental retardation of the fetus.

The strict low-phenylalanine regimen is tiresome for the patients andtheir families, and is very difficult to enforce beyond childhood.Enzyme therapy to make up for the phenylalanine hydroxylase deficiencywould therefore provide a great improvement in the treatment ofhyperphenylalaninemia. Unlike phenylalanine hydroxylase, anotherphenylalaninedegrading enzyme, phenylalanine ammonia lyase, requires nocofactors to be active. Phenylalanine ammonia lyase convertsphenylalanine to trans-cinnamic acid which, via coenzyme A, is convertedto benzoic acid which reacts with glycine and is then excreted via urineprimarily as hippurate. The enzyme may, for instance, be obtained fromthe yeast Rhodotorula glutinis (also known as Rhodosioridiumtoruloides). It has previously been suggested to use phenylalanineammonia lyase for treatment of hyperphenylalaninemia, vide for instance,J. A. Hoskins et al., Lancet, Feb. 23, 1980, pp. 392-394. Proteolyticdegradation of the enzyme in the gastrointestinal tract has beenrecognized, e.g. by H. J. Gilbert and G. W. Jack, Biochem. J. 199, 1981,pp. 715-723. Various attempts to overcome this problem have beenpublished. Thus, L. Bourget and T. M. S. Chang, Biochim. Biophys. Acta883, 1986, pp. 432-438, propose microencapsulation of the enzyme in"artificial cells" composed of phenylalanine ammonia lyase mixed withhemoglobin and enclosed in microspheres covered by a cellulose nitratemembrane. H. J. Gilbert and M. Tully, Biochem. Biophys. Res. Comm.131(2), 1985, pp. 557-563 propose using permeabilised cells ofRhodosporidium toruloides containing the enzyme. However, both of theseapproaches may have drawbacks such as low specific phenylalanine ammonialyase activity of the final preparation or high cost due to processingor formulation.

DESCRIPTION OF THE INVENTION

The object of the present invention is to overcome the drawbacks of thepreviously suggested methods of stabilising phenylalanine ammonia lyase.

Accordingly, the present invention relates to an enzyme preparationcomprising phenylalanine ammonia lyase (PAL) stabilised againstproteolytic degradation by chemical or genetic modification.

In a preferred embodiment of the enzyme preparation of the invention,the PAL is chemically stabilised by treatment with a cross-linkingagent. For the present purpose, it is assumed that the cross-linkingagent reinforces the conformation of PAL and makes it less accessible toproteolytic enzymes in the gastrointestinal tract by reticulating themolecule (intramolecular cross-links) to form a brace. Intermolecularcross-linking to another molecule (typically another protein) may alsobe advantageous to form a conjugate in which the enzyme is protectedfrom the action of proteases. A further description of chemicalcross-linking of proteins may be found in, e.g., S. S. Wong and L.-J. C.Wong, Enzyme Microb. Technol. 14, 1992, pp. 866-873. Preferredcross-linking reagents are selected from aldehydes, isocyanates,isothiocyanates, anhydrides and azides. Particularly preferredcross-linking agents are bifunctional reagents, i.e. compounds with tworeactive groups. Examples of such reagents are pharmaceuticallyacceptable carbodiimides, isoxazolium derivatives, chloroformates,carbonyldiimidazole, bis-imidoesters, bis-succinimidyl derivatives,di-isocyanates, di-isothiocyanates, disylfonyl halides, bis-nitrophenylesters, dialdehydes, diacylazides, bis-maleimides, bis-haloacetylderivatives, di-alkyl halides and bis-oxiranes. A currently preferredcross-linking agent for the present purpose is glutaraldehyde. Thiscompound is inexpensive, readily available and approved for a number offood-related enzyme applications. It yields a product with goodmechanical properties and good recovery of the enzymatic activity.

The PAL is an intracellular enzyme and may as such be present in wholecells or permeabilised cells, or it may be present in a cell homogenate.The PAL may also be cell-free, affording a preparation which is notdiluted with enzymatically inactive cell material and consequently isenriched in enzymatic activity. In the enzyme preparation of theinvention, PAL preferably constitutes at least 25%, in particular atleast 50%, of the enzyme protein in the preparation. In a specificembodiment, the PAL is in crystalline (i.e. substantially pure) formwhich may be advantageous for formulation, dosage or approval purposes.

The cross-linking reaction may be carried out at room temperature or atlower temperatures. Higher reaction temperatures during cross-linkingmay inactivate the enzyme. For effective cross-linking, the reactiontime may vary from a few minutes to several hours. The pH of thecross-linking medium should be one which ensures reactivity of thecross-linking agent concomitantly with enzyme activity. Whenglutaraldehyde is used as the cross-linking agent, a pH of about 6-10will be the most appropriate. In case of cell-free or crystalline PAL,it may be advantageous to include an auxiliary substance such as apolyamine in the cross-linking reaction.

In another aspect, the present invention relates to a PAL variantstabilised against proteolytic degradation, wherein one or more aminoacid residues susceptible to proteolytic cleavage are substituted by oneor more amino acid residues less susceptible to proteolytic cleavage.

In the present description and claims, the following abbreviations areused:

Amino Acids

A=Ala=Alanine

V=Val=Valine

L=Leu=Leucine

I=Ile=Isoleucine

P=Pro=Proline

F=Phe=Phenylalanine

W=Trp=Tryptophan

M=Met=Methionine

G=Gly=Glycine

S=Ser=Serine

T=Thr=Threonine

C=Cys=Cysteine

Y=Tyr=Tyrosine

N=Asn=Asparagine

Q=Gln=Glutamine

D=Asp=Aspartic Acid

E=Glu=Glutamic Acid

K=Lys=Lysine

R=Arg=Arginine

H=His=Histidine

In describing PAL variants according to the invention, the followingnomenclature is used for ease of reference: Original amino acid(s)position(s) substituted amino acid(s)

According to this nomenclature, for instance the substitution of alaninefor phenylalanine in position 629 is shown as:

    F629A

According to the invention, it has been found that the amino acidresidues Phe, Tyr, Trp, Lys and Arg are particularly sensitive tocleavage by the major proteolytic enzymes in the gastrointestinal tract,i.e. chymotrypsin (cleavage at Phe, Tyr and Trp) and trypsin (cleavageat Lys and Arg). To improve the stability of PAL in the gastrointestinaltract, one or more of these amino acid residues may therefore bereplaced by other residues which are more resistant to proteolyticcleavage.

The parent PAL may be derivable from a microorganism, in particular afungus such as a Rhodotorula sp., Rhodos-oridium sp., Sporobolus sp.,Geotrichum sp., Moniliella sp., Pellicularia sp, Gonatobotryum sp.,Syncerhalastrum sp., Endomyces sp., Aspergillus sp., Saccharomvcopsissp., Eurotium sp., Glomerella sp., Cladosporium sp. or Trichosporon sp.,or from a plant such as Pisum sativum, potato, sweet potato or soy bean.A particularly preferred PAL is one derivable from a strain ofRhodosporidium toruloides (syn. Rhodotorula glutinis), or a suitablehomologue thereof.

In the present context, the term "homologue" is intended to indicate aPAL of which the amino acid sequence is at least 45% identical to thatof the Rhodosporidium toruloides PAL. Sequence comparisons may beperformed via known algorithms, such as the one described by Lipman andPearson, Science 227, 1985, p. 1435. Sequences may be obtained fromdatabases containing Published Sequences. Examples of homologues arePALs derivable from Rhodotorula rubra, Lycopersicon esculentum,Nicotiana tabacum, Ipomoea batatas, Phaseolus vulgaris, Medicago sativa,Petroselinum crispum, Oryza sativa and soybean.

In particular, the protease-stability of PAL may be improved bysubstituting one or more amino acid residues in the region from aminoacid 629 to 674 of the PAL derivable from Rhodosporidium toruloides.Without wishing to be limited to any theory, it is currently assumedthat this region forms a loop on the surface of the enzyme, so that theprotease-sensitive amino acid residues present in this region areparticularly exposed to proteolytic enzymes in the gastrointestinaltract. It is anticipated that amino acid residues in correspondingpositions of homologous PALs may likewise be substituted.

More specifically, one or more amino acid residues may be substituted asfollows

F629A,S,V,L,E,P,N,I,Q,T,M,G,H,D

F631A,S,V,L,E,P,N,I,Q,T,M,G,H,D

W653A,S,V,L,E,P,N,I,Q,T,M,G,H,D

K654A,S,V,L,E,P,N,I,Q,T,M,G,H,D

R667A,S,V,L,E,P,N,I,Q,T,M,G,H,D

R670A,S,V,L,E,P,N,I,Q,T,M,G,H,D

F673A,S,V,L,E,P,N,I,Q,T,M,G,H,D

W674A,S,V,L,E,P,N,I,Q,T,M,G,H,D

Cloning a DNA Sequence Encoding a PAL

The DNA sequence encoding a parent PAL may be isolated from any cell ormicroorganism producing the PAL in question by various methods, wellknown in the art. Firstly, a genomic DNA and/or cDNA library should beconstructed using chromosomal DNA or messenger RNA from the organismthat produces the PAL to be studied. Then, if the amino acid sequence ofthe PAL is known, homologous, labelled oligonucleotide probes may besynthesized and used to identify PAL-encoding clones from a genomiclibrary of bacterial DNA, or from a fungal cDNA library. Alternatively,a labelled oligonucleotide probe containing sequences homologous to PALfrom another strain of bacteria or fungus could be used as a probe toidentify PAL-encoding clones, using hybridization and washing conditionsof lower stringency.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers, TetrahedronLetters 22, 1981, pp. 1859-1869, or the method described by Matthes etal., The EMBO J. 3, 1984, pp. 801-805. According to the phosphoamiditemethod, oligonucleotides are synthesized, e.g. in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Finally, the DNA sequence may be of mixed genomic and synthetic, mixedsynthetic and cDNA or mixed genomic and cDNA origin prepared by ligatingfragments of synthetic, genomic or cDNA origin (as appropriate), thefragments corresponding to various parts of the entire DNA sequence, inaccordance with standard techniques. The DNA sequence may also beprepared by polymerase chain reaction (PCR) using specific primers, forinstance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al.,Science 239, 1988, pp. 487-491.

Site-directed Mutagenesis of the PAL-encoding Sequence

Once a PAL-encoding DNA sequence has been isolated, and desirable sitefor mutation identified, mutations may be introduced using syntheticoligonucleotides. These oligonucleotides contain nucleotide sequencesflanking the desired mutation sites; mutant nucleotides are insertedduring oligonucleotide synthesis. In a specific method, asingle-stranded gap of DNA, bridging the PAL-encoding sequence, iscreated in a vector carrying the PAL gene. Then the syntheticnucleotide, bearing the desired mutation, is annealed to a homologousportion of the single-stranded DNA. The remaining gap is then filled inwith DNA polymerase I (Klenow fragment) and the construct is ligatedusing T4 ligase. A specific example of this method is described inMorinaga et al., (1984, Biotechnology 2:646-639). U.S. Pat. No.4,760,025, by Estell et al., issued Jul. 26, 1988, discloses theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette, however, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

Another method of introducing mutations into PAL-encoding sequences isdescribed in Nelson and Long, Analytical Biochemistry 180, 1989, pp.147-151. It involves the 3-step generation of a PCR fragment containingthe desired mutation introduced by using a chemically synthesized DNAstrand as one of the primers in the PCR reactions. From thePCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intothe plasmid.

Expression of PAL Variants

According to the invention, a mutated PAL-encoding sequence produced bymethods described above, or any alternative methods known in the art,may be expressed using an expression vector which typically includescontrol sequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes. To permit secretion of the expressed protein,nucleotides encoding a "signal sequence" may be inserted prior to thePAL-coding sequence. For expression under the direction of controlsequences, a target gene to be treated according to the invention isoperably linked to the control sequences in the proper reading frame.Promoter sequences that can be incorporated into plasmid vectors, andwhich can support the transcription of the mutant PAL gene, include butare not limited to the prokaryotic β-lactamase promoter (Villa-Kamaroff,et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731) and the tacpromoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25).Further references can also be found in "Useful proteins fromrecombinant bacteria" in Scientific American, 1980, 242:74-94.

According to one embodiment B. subtilis is transformed by an expressionvector carrying the mutated DNA. If expression is to take place in asecreting microorganism such as B. subtilis a signal sequence may followthe translation initiation signal and precede the DNA sequence ofinterest. The signal sequence acts to transport the expression productto the cell wall where it is cleaved from the product upon secretion.The term "control sequences" as defined above is intended to include asignal sequence, when present.

In a currently preferred method, the PAL or PAL variants may be producedin a yeast host cell expressing a DNA sequence encoding the enzyme.Examples of preferred yeast hosts are Saccharomyces e.g. Saccharomycescerevisiae or Saccharomyces kluyveri, Schizosaccharomyces, e.g.Schizosaccharomyces pombe, Kluvveromyces, e.g. Kluyveromyces lactis,Pichia, e.g. Pichia pastoris, or Yarrowia, e.g. Yarrowia lipolvtica. ThePAL may also be produced in Rhodosporidium toruloides from which thegene is preferentially derived.

The DNA sequence encoding PAL may, for instance be isolated as describedin GB 2 213 486. As the amino acid sequence of PAL is known, it may alsobe possible to construct a synthetic gene encoding the enzyme.

The intracellular expression of PAL may be achieved by linking thePAL-encoding DNA sequence to a suitable control system such as apromoter, ribosome-binding sequences and terminator sequence. Suitablepromoters for use in yeast host cells include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255, 1980, pp.12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp.419-434) or alcohol dehydrogenase genes (Young et al., in GeneticEngineering of Microorganisms for Chemicals (Hollaender et al, eds.),Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No. 4,599,311) orADH2-4c (Russell et al., Nature 304, 1983, pp. 652-654) promoters.

The procedures used to ligate the DNA sequences coding for the PAL, thepromoter and the terminator, respectively, and to insert them intosuitable vectors containing the information necessary for replication,are well known to persons skilled in the art (cf., for instance,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989).

To provide extracellular production of PAL into the culture medium, thecontrol system may include a suitable signal sequence, such as the MFasignal/leader sequence (Kurjan and Herskowitz, Cell, 1982, pp. 933-943).Examples of other signal/leader sequences are described in WO 89/02463and WO 92/11378.

Transformation of the yeast cells with a vector containing the PAL geneand expression thereof may be carried out according to well-knownprocedures, e.g. as described in WO 90/10075.

In another method of producing PAL or PAL variants of the invention, afilamentous fungus is used as the host organism. The filamentous fungushost organism may conveniently be one which has previously been used asa host for producing recombinant proteins, e.g. a strain of Aspercillussp., such as A. niger, A. nidulans or A. oryzae. The use of A. oryzae inthe production of recombinant proteins is extensively described in, e.g.EP 238 023.

For expression of PAL variants in Asperaillus, the DNA sequence codingfor the PAL variant is preceded by a promoter. The promoter may be anyDNA sequence exhibiting a strong transcriptional activity in Asperaillusand may be derived from a gene encoding an extracellular orintracellular protein such as an amylase, a glucoamylase, a protease, alipase or a glycolytic enzyme.

Examples of suitable promoters are those derived from the gene encodingA. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease orA. oryzae triose phosphate isomerase.

In particular when the host organism is A. oryzae, a preferred promoterfor use in the process of the present invention is the A. oryzae TAKAamylase promoter as it exhibits a strong transcriptional activity in A.oryzae. The sequence of the TAKA amylase promoter appears from EP 238023.

Termination and polyadenylation sequences may suitably be derived fromthe same sources as the promoter.

The techniques used to transform a fungal host cell may suitably be asdescribed in EP 238 023.

To ensure secretion of the PAL variant from the host cell, the DNAsequence encoding the PAL variant may be preceded by a signal sequencewhich may be a naturally occurring signal sequence or a functional partthereof or a synthetic sequence providing secretion of the protein fromthe cell. In particular, the signal sequence may be derived from a geneencoding an Aspergillus sp. amylase or glucoamylase, a gene encoding aRhizomucor miehei lipase or protease, or a gene encoding a Humicolacellulase, xylanase or lipase. The signal sequence is preferably derivedfrom the gene encoding A. oryzae TAKA amylase, A. niger neutralα-amylase, A. niger acid-stable α-amylase or A. niger glucoamylase.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing Asperaillus cells. Thetransformants are usually stable and may be cultured in the absence ofselection pressure. However, if the transformants are found to beunstable, a selection marker introduced into the cells may be used forselection.

The mature PAL protein may conveniently be recovered from the culture bywell-known procedures including lysing the cells and precipitatingproteinaceous components of the medium by means of a salt such asammonium sulphate, followed by chromatographic procedures such as ionexchange chromatography, affinity chromatography, or the like.

The present invention also relates to a pharmaceutical compositioncontaining the enzyme preparation or PAL variant of the inventiontogether with a pharmaceutically acceptable carrier or excipient. In thecomposition of the invention, the enzyme may be formulated by any one ofthe established methods of formulating pharmaceutical compositions, e.g.as described in Remington's Pharmaceutical Sciences, 1985. Thecomposition should be in a form adapted for oral administration,including a powder, granulate, tablet, capsules, microcapsule, solutionor suspension. Suitable carriers and excipients for oral administrationare well known in the art.

The pharmaceutical composition of the invention is suitably provided inunit dosage form such as a tablet or capsule. To protect the enzyme fromdegradation by gastric fluid, such tablets or capsules are preferablyprovided with an enteric coating, that is, a coating which is insolubleat gastric pH but dissolves at intestinal pH (typically at a pH of 5 ormore). Examples of suitable enteric coating agents are cellulose acetatephthalate (CAP, Cellacephate®), vinyl acetate crotonic acid copolymer(Luviset®), methacrylic acid, (meth)acrylic acid ester copolymer(Eudragit®) or hydroxypropyl methylcellulose phthalate. For a furtherdescription of enteric coatings and coating processes, reference is madeto WO 87/07292. Another suitable pharmaceutical composition is acontrolled release formulation from which the enzyme is released duringits passage through the gastrointestinal tract.

The composition of the invention may be used for the prevention ortreatment of hyperphenylalaninemia, in particular phenylketonuria, aspreviously suggested by i.a. J. A. Hoskins et al., Lancet, Feb. 23,1980, pp. 392-394; H. J. Gilbert and M. Tully, Biochem. Biophys. Res.Comm. 131(2), 1985, pp. 557-563; and L. Bourget and T. M. S. Chang,Biochim. Biophys. Acta 883, 1986, pp. 432-438. A suitable dose of PAL tokeep the plasma phenylalanine level below the critical level is in therange of from about 50 to about 500 mg PAL protein per day, inparticular about 200 mg PAL protein per day.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

Example 1

Cultivation of PAL Producing Cells

Four strains: Rhodotorula craminis (ATCC 20804), Rhodotorula minuta(NRRL Y-1589), Rhodosporidium toruloides (NRRL Y-1091), and Rhodotorulaaurantiaca (NRRL Y-7219) were cultured in shake flasks at the followingconditions:

Medium: 2% yeast extract, 2% peptone, 0.4% phenylalanine, and 6% glucose

Culture: 26° C., 250 rpm, 4 days

PAL activity was analysed on supernatants after homogenization ofculture broth with 1 g of glass beads pr. ml.

Permeabilisation and Immobilisation of Cells by GlutaraldehydeModification

Cells were diluted with water to reduce viscosity and adjusted to pH 8.They were then frozen at -20° C. in ethanol/dry ice and thawed forpermeabilisation. During ice bath cooling, glutaraldehyde was added to afinal concentration of 0.5%. pH was readjusted to 8 and stirringcontinued for 1 hour. The cells were centrifuged for 10 minutes at 7000rpm. This was repeated after decantation and suspension in 20 ml waterfollowed by freeze-drying of the cells. Details regarding the three cellbatches are given below:

    ______________________________________                                        Strain/batch                                                                           ATCC 20804  NRRL Y-1589 ATCC 20804                                   ______________________________________                                        Cell activity                                                                          --          --          6.6 U/ml*                                    Cell volume                                                                            11 ml       16 ml       9.3 g                                        used                                                                          Added water                                                                            9 ml        2 ml        8.2 g                                        pH       5.6         6.2         5.7                                          4N NaOH →                                                                       400 μl   400 μl   200 μl                                    pH 8                                                                          Glutaralde-                                                                            200 μl   180 μl   177 μl                                    hyde, 50%                                                                     Immobilized                                                                            1.13 g      2.15 g      0.94 g                                       PAL                                                                           Activity 0.57 U/g    1.96 U/g    11.3 U/g                                     ______________________________________                                         (#) Immobilization: 2 hours at room temperature. Vacuum drying overnight      at 40° C. Ground in mortar.                                            (*) Approx. activity based on concentrated culture broth.                

Comparison Preparations

Acetone permeabilised cells were made basically as described by Gilbertand Tully. The cells were diluted with water and adjusted to pH 8 as forthe glutaraldehyde treated cells. They were then added dropwise to 20volumes of -10° C. acetone (made with dry ice). After standing 15minutes under occasional stirring, they were allowed to settle for 5-10minutes. After filtering and re-suspension in 200 ml cold water, cellswere collected by centrifugation, 7000 rpm for 12 minutes, followed byfreeze-drying. Details for the two batches are given below:

    ______________________________________                                        Strain/batch   NRRL Y-1091  NRRL Y-7219                                       ______________________________________                                        Cell volume used                                                                             13 ml        18 ml                                             Added water    8 ml         7 ml                                              pH             7.2          7.7                                               4N NaOH → pH 8                                                                        100 μl    100 μl                                         Acetone        200 ml       240 ml                                            Dry cell yield 0.32 g       0.44 g                                            Activity       1.23 U/g     1.79 U/g                                          ______________________________________                                    

Activity and Stability Analyses

PAL activity was determined in principle by the measurement of cinnamateat 290 nm from 13 mM phenylalanine, 0.1M Tris, pH 8.5, 37° C. describedby Gilbert and Jack. A linear relation between cinnamate concentrationand OD₂₉₀ allows calculation of PAL activity in units of μmolescinnamate/-minute. In order to assay the immobilized preparations,incubation of samples was followed by separation of immobilized PALbefore measurement at 290 nm.

Stability towards chymotrypsin with subsequent assay of residual PALactivity was analyzed as follows:

Chymotrypsin (Novo Nordisk, 1000 USP/mg, <25 USP/mg trypsin) was addedto the 13 mM phenylalanine substrate (0.1M Tris, pH 8.5) inconcentrations of 0, 3, 30, and 300 USP/ml. 25 mg immobilized PAL wasadded to 10 ml substrate, preheated to 37° C., and shaken at 37° C.(vertically in 20 ml glass tubes). 3.0 ml samples were taken at 5 and 30minutes and filtered before OD₂₉₀ measurement. Reference: Substrate withchlymotrypsin. Blank samples: Tris-buffer without phenylalanine, withchymotrypsin, and with immobilized PAL. Reference to blank samples:Chymotrypsin in Tris-buffer.

Activity calculation:

    Activity in U/g=ε*1*v* (E.sub.30 -E.sub.30B)-(E.sub.5 -E.sub.5B)!/t*m

ε=molar extinction coefficient of cinnamate: 1*10⁴ liter/-mole/cm

1=cuvette width: 1 cm

V=substrate volume: 10 ml

t=reaction time: 25 minutes

m=amount of enzyme: 25 mg

E₃₀ =extinction of sample at 30 minutes

E_(30B) =extinction of blank at 30 minutes

E₅ =extinction of sample at 5 minutes

E_(5B) =extinction of blank at 5 minutes

With these values, A=1.60* (E₃₀ -E_(30B))-(E₅ -E_(5B))!, in U/g

Soluble PAL (Sigma P-1016, lot 22H8000, from Rhodotorula glutinis) wasanalysed to 2.8 U/ml and diluted 1:10 prior to incubation with 2.6USP/ml chymotrypsin (batch as above) in 0.1M Tris, pH 7.5, 370° C.Residual PAL activity was assayed after 15 and 30 minutes and calculatedrelative to original activity.

    ______________________________________                                        Results:                                                                                Residual activity, U/g and %,                                       Immobilized                                                                             at different chymotrypsin levels                                    preparation                                                                             0 USP/ml 3 USP/ml  30 USP/ml                                                                             300 USP/ml                               ______________________________________                                        Glutaraldehyde                                                                          0.57     0.53      0.53    0.44                                     treated   100      93        93      77                                       ATCC 20804100                                                                 Glutaraldehyde                                                                          1.96     1.69      1.76    1.34                                     treated   100      86        90      68                                       NRRL Y-1589                                                                   Glutaraldehyde                                                                          11.3     --        --      11.3                                     treated   100      --        --      100                                      ATCC 20804                                                                    Comparisons                                                                   preparations                                                                  Acetone permea-                                                                         1.23     0.89      0.55    0.28                                     bilized   100      72        45      23                                       NRR1 Y-1091                                                                   Acetone permea-                                                                         1.79     1.11      0.90    0.54                                     bilized   100      62        50      30                                       NRRL Y-7219                                                                   Soluble PAL                                                                             2.8 U/ml 0.02 U/ml                                                          100%   0.8% (2.5% after 15 minutes)                                   ______________________________________                                    

These results demonstrate the efficiency of glutaraldehyde treatment instabilizing PAL-containing Rhodotorula cells towards chymotrypsin.

We claim:
 1. An enzyme preparation comprising phenylalanine ammonialyase (PAL) stabilised against proteolytic degradation by treatment witha cross-linking agent which is a bifunctional reagent or in which one ormore amino acid residues susceptible to proteolytic cleavage arereplaced by other amino acid residues less susceptible to proteolyticcleavage.
 2. The enzyme preparation according to claim 1 which includeswhole cells containing PAL, permeabilised cells containing PAL, a cellhomogenate containing PAL or cell-free PAL.
 3. The enzyme preparationaccording to claim 1, in which PAL constitutes at least 25% of theenzyme protein in the preparation.
 4. The enzyme preparation accordingto claim 1, in which PAL constitutes at least 50% of the enzyme proteinin the preparation.
 5. The enzyme preparation according to claim 1,wherein the PAL is in crystalline form.
 6. The enzyme preparationaccording to claim 1, wherein the cross-linking agent is selected fromthe group consisting of pharmaceutically acceptable carbodiimides,isoxazolium derivatives, chloroformates, carbonyldiimidazole,bis-imidoesters, bis-succinimidyl derivatives, di-isocyanates,di-isothiocyanates, di-sylfonyl halides, bis-nitrophenyl esters,dialdehydes, diacylazides, bis-maleimides, bis-haloacetyl derivatives,di-alkyl halides and bis-oxiranes.
 7. The enzyme preparation accordingto claim 4, wherein the dialdehyde is glutaraldehyde.
 8. The enzymepreparation according to claim 1, wherein the PAL is derivable from amicroorganism.
 9. The enzyme preparation according to claim 1, whereinthe PAL is derivable from a fungus.
 10. The enzyme preparation accordingto claim 1, wherein the PAL is derivable from a fungus selected from thegroup consisting of Rhodotorula sp., Rhodosporidium sp., Sporobolus sp.,Geotrichum sp., Moniliella sp., Pellicularia sp, Gonatobotryum sp.,Syncephalastrum sp., Endomyces sp., Aspergillus sp., Saccharomycopsissp., Eurotium sp., Glomerella sp., Cladosporium sp. and Trichosporon sp.11. The enzyme preparation according to claim 1, wherein the PAL isderivable from a plant.
 12. The enzyme preparation according to claim 1,wherein the PAL is derivable from a plant selected from the groupconsisting of Pisum sativum, potato, sweet potato and soy bean.
 13. APAL variant stabilised against proteolytic degradation, wherein one ormore amino acid residues susceptible to proteolytic cleavage arereplaced by other amino acid residues less susceptible to proteolyticcleavage.
 14. The PAL variant according to claim 13, wherein one or moreof the amino acid residues Phe, Tyr, Trp, Lys or Arg are replaced byother amino acid residues.
 15. The PAL variant according to claim 13,wherein the parental PAL is derived from a microorganism.
 16. The PALvariant according to claim 13, wherein the parental PAL is derived froma fungus.
 17. The PAL variant according to claim 13, wherein theparental PAL is derived from a fungus selected from the group consistingof Rhodotorula sp., Rhodosporidium sp., Sporobolus sp., Geotrichum sp.,Moniliella sp., Pellicularia sp, Gonatobotryum sp., Syncephalastrum sp.,Endomyces sp., Aspergillus sp., Saccharomycopsis sp., Eurotium sp.,Glomerella sp., Cladosporium sp. and Trichosporon sp.
 18. The PALvariant according to claim 13, wherein the parental PAL is derived froma plant.
 19. The PAL variant according to claim 13, wherein the parentalPAL is derived from a plant selected from the group consisting of Pisumsativum, potato, sweet potato and soy bean.
 20. The PAL variantaccording to claim 13, wherein the parental PAL is derived fromRhodosporidium toruloides.
 21. The PAL variant according to claim 20,wherein one or more amino acid residues are substituted in the regionfrom amino acid 629 to
 674. 22. The PAL variant according to claim 21,wherein one or more amino acid residues are substituted asfollowsF629A,S,V,L,E,P,N,I,Q,T,M,G,H,D F631 A,S,V,L,E,P,N,I,Q,T,M,G,H,DW653A,S,V,L,E,P,N,I,Q,T,M,G,H,D K654A,S,V,L,E,P,N,I,Q,T,M,G,H,DR667A,S,V,L,E,P,N,I,Q,T,M,G,H,D R670A,S,V,L,E,P,N,I,Q,T,M,G,H,DF673A,S,V,L,E,P,N,I,Q,T,M,G,H,D W674A,S,V,L,E,P,N,I,Q,T,M,G,H,D.
 23. ADNA construct comprising a DNA sequence encoding the PAL variantaccording to claim
 13. 24. A recombinant expression vector comprising aDNA construct according to claim
 23. 25. A cell transformed with a DNAconstruct according to claim
 23. 26. The cell according to claim 25, inwhich the cell is a microbial cell.
 27. The cell according to claim 25,in which the cell is a yeast cell.
 28. The cell according to claim 25,in which the cell is a yeast cell selected from the group consisting ofRhodotorula glutinis, Saccharomyces, Schizosaccharomyces, Kluyveromyces,Pichia, and Yarrowia.
 29. The cell according to claim 25, in which thecell is a Saccharomyces cerevisiae or Saccharomyces kluyveri cell. 30.The cell according to claim 25, in which the cell is a Kluyveromyceslactis cell.
 31. The cell according to claim 25, in which the cell is aYarrowia lipolytica cell.
 32. The cell according to claim 23, in whichthe cell is a Schizosaccharomyces pombe cell.
 33. The cell according toclaim 25, in which the cell is a Pichia pastoris cell.
 34. The cellaccording to claim 25, in which the cell is a filamentous fungal cell.35. The cell according to claim 25, in which the cell is a filamentousfungal cell selected from the group consisting of Aspergillus andTrichoderma.
 36. The cell according to claim 25, in which the cell isselected from the group consisting of Aspergillus niger, Aspergillusoryzae and Aspergillus nidulans.
 37. The cell according to claim 25, inwhich the cell is a Trichoderma reseei cell.
 38. A process for preparinga PAL variant according to claim 13, comprising culturing a celltransformed with a DNA construct comprising a DNA sequence encoding thePAL variant in a suitable culture medium under conditions permittingproduction of the PAL variant, and recovering the resulting PAL variantfrom the culture.
 39. A pharmaceutical composition adapted for oraladministration comprising an enzyme preparation according to claim 1together with a pharmaceutically acceptable carrier or excipient.
 40. Apharmaceutical composition for the prevention or treatment ofhyperphenylalaninemia comprising an enzyme preparation according toclaim 1 together with a pharmaceutically acceptable carrier orexcipient.
 41. A composition according to claim 40 in the form of atablet or capsule provided with an enteric coating.
 42. A pharmaceuticalcomposition adapted for oral administration comprising a PAL variantaccording to claim 13 together with a pharmaceutically acceptablecarrier or excipient.
 43. A pharmaceutical composition for theprevention or treatment of hyperphenylalaninemia comprising a PALvariant according to claim 13 together with a pharmaceuticallyacceptable carrier or excipient.
 44. A composition according to claim 42in the form of a tablet or capsule provided with an enteric coating.